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Abstract

Light emitting diodes (LED), which are available as small monochromatic light sources
with characteristic features such as maximum illumination power combined with minimum
energy consumption and extremely long lifespan have already proved as a highly potential
low-cost alternative for specific diagnostic applications in clinical medicine such
as tuberculosis fluorescence microscopy. Likewise, the most reliable evaluation of
Her-2/neu (c-erbB2) gene amplification, which has been established in the last few
years for routine diagnosis in clinical pathology as determinant towards Herceptin-based
treatment of patients with breast cancer, is based on fluorescence in situ hybridization (FISH) and corresponding high priced fluorescence equipment. In order
to test the possibility to utilize the advantages of low-cost LED technology on FISH
analysis of c-erbB2 gene expression for routine diagnostic purposes, the applicability
of a standard bright field Carl Zeiss Axiostar Plus microscope equipped with a Fraen
AFTER* LED Fluorescence Microscope Kit for the detection of Her-2/neu gene signals
was compared to an advanced Nikon Eclipse 80i fluorescence microscope in combination
with a conventional 100W mercury vapor lamp. Both microscopes were fitted with the
same Quicam FAST CCD digital camera to unequivocally compare the quality of the captured
images. C-erbB2 gene expression was analyzed in 30 different human tissue samples
of primary invasive breast cancer, following formalin fixation and subsequent paraffin-embedding.
The Her2/neu gene signals (green) were identifiable in the tumor cells in all cases
and images of equal quality were captured under almost identical conditions by 480
nm (blue) LED module equipped standard Axiostar microscope as compared to conventional
fluorescence microscopy. In this first attempt, these monochromatic LED elements proved
in principle to be suitable for the detection of Her-2/neu gene expression by FISH.
Thus, our own experiences emphasize the high potential of this technology to provide
a serious alternative to conventional fluorescence microscopy in routine pathology;
representing a sustainable technological progress, this low-cost technology will clearly
give direction also to the growing field of molecular pathology.

* AFTER = Amplified Fluorescence by transmitted Excitation of Radiation

Findings

Light Emitting Diodes (LED) are characterized by low cost, effective energy consumption
and extremely long lifespan when compared to conventional light sources [1]. Both the small size and the lack of heat development by LED elements also contribute
to these major advantages above conventional lighting technology that altogether imply
considerable economic cost reductions. As a consequence, there has been a fast propagation
of LED elements as important constituents in many different branches such as automobile
industry, household or camping [2,3]. More recently, the availability of a variety of small monochromatic LED modules
efficiently emitting the spectrum in a single desired bandwidth has stimulated the
interest of clinical researchers to utilize this low-cost technology for advanced
fluorescence microscopy in diagnostic research [4]. Since LED based light sources operate without increasing their temperature, common
safety problems related to considerable heat production by conventional high pressure
mercury lamps are completely avoided. Most recently, LED modules attached to standard
light microscopes have been successfully applied in fluorescence-based screening of
tuberculosis, pointing to the considerable reduction of related costs combined with
increased safety and, as a consequence, to the potential for low-income countries
to perform such advanced diagnostics of this disease in the near future [5,6]. Likewise, the increasing application of highly sensitive but also expensive molecular
methods in clinical pathology such as the analysis of altered gene expression patterns
for cancer diagnosis, has led to a general demand for reducing costs in routine processes.
In the last few years, the evaluation of Her-2/neu status has considerably gained
clinical importance related to the selection of patients with breast cancer, who will
benefit most from a novel targeted therapy based on Herceptin, a humanized monoclonal
antibody directed against this protein [7,8]. Thus, overexpression of Her-2/neu protein represents one of only few available predictive
markers for an individualized and more efficient treatment regimen in this type of
cancer [9]. Since the enhancement of protein levels is primarily correlated to the amplification
of the corresponding gene c-erbB2, fluorescence in situ hybridization (FISH) has been established for the determination of Her-2/neu gene.
FISH is characterized by excellent sensitivity (96.5%) and specificity (100%) [10]. Thus, this diagnostic assay has been introduced in routine clinical pathology, despite
the considerable costs involving expensive reagents and the need to purchase high
priced fluorescent equipment before deciding the further steps in the comparably much
more expensive treatment with all its possible therapeutic side effects 1.

For this reason, the use of small monochromatic LED modules as the required light
source for routinely performed FISH analysis of Her-2/neu status appeared to us as
a promising alternative to eventually replace the short-lived and expensive conventional
mercury vapor lamp. For this purpose, a commercially available AFTER (Amplified Fluorescence
by transmitted Excitation of Radiation) LED Fluorescence Microscope Kit (Lab Vision,
Fremont, USA) was mounted to a standard Zeiss Axiostar Plus transmitted light microscope
(Medac, Wedel, Germany), providing a simple adaptation of a fluorescence microscope.
Determination of ZyGreen c-erb-B2 gene was performed by attaching a 480 nm Fraen fluorescence
light cassette to the Axiostar Plus in combination with a LP 510 nm long pass filter.
For comparison reasons, the Zeiss microscope was also fitted with a Quicam FAST CCD
digital camera, which is normally attached to the Nikon Eclipse 80i fluorescence microscope
for routine determination of cerb-B2 gene amplification. Her-2/neu gene expression
was documented at 400× magnification. A total of 30 tissue samples from patients with
primary invasive breast carcinoma were analyzed by FISH. After mastectomy, the specimens
were fixed by formalin and subsequently paraffin-embedded. Optimal comparability among
all samples was achieved by producing a tissue micro array (TMA), as previously described
[11]. FISH was performed, using ZytoLight Spec Her-2 Color Probe (ZytoVision GmbH, Bremerhaven,
Germany). Briefly, a 4 μm thick section of the TMA was deparaffinized with xylene,
dehydrated and pre-treated with enzyme and heat, according to the manufacturer's instructions.
After addition of 10 μl SPEC HER2 Color probe, denaturation was carried out at 75°C
for 10 min, followed by hybridization overnight at 37°C. Post hybridization washing,
subsequent dehydration in ethanol and counterstaining with DAPI (4,6-diaminido-2-phenylindole
dihydrochloride)/antifade-solution was performed, as specified. The slides were kept
in the dark at 4°C until evaluation.

The age of the patients ranged between 44 years and 84 years (median age 61 y) with
77% of the women being in the postmenopausal phase. The majority of the breast cancer
tissues (86%) were related histologically to the invasive ductal type of tumor. Amplification
of cerb-B2 gene was detected by FISH in 36% of the specimens. Brightness of the Her-2/neu
gene signals was always sufficient using the LED equipped Axiostar Plus microscope
and corresponding photographs of high quality were captured by extended integration
times between 3 and 10 seconds.

To our knowledge, this is the first attempt to use LED modules instead of a 100 W
mercury vapor lamp as light source to perform FISH analysis of clinically relevant
Her-2/neu gene expression for routine pathology. Up to date, the introduction of LED
technology in diagnostic research has been very successful as recently demonstrated
for the fluorescence based screening of tuberculosis [5,6,12]. Likewise, in our studies the standard light transmission microscope Axiostar Plus
became suitable for FISH analysis by simply attaching a commercially available adaptation
kit for fluorescence microscopy. The appropriate combination of a particular LED module
and the corresponding long pass emission filter was sufficient to replace the advanced
Nikon fluorescence microscope. Moreover, the lack of heat production by the LED light
sources completely avoided common safety problems related to conventional mercury
vapor lamps. The extension of the integration time was the only major modification
of the otherwise identical conditions to capture pictures of equal quality as compared
to the routinely used high priced fluorescence equipment. Integration times less than
3 times shorter to be sufficient for documentation, as demonstrated for the 100 W
mercury lamp, still emphasize the need for LED modules with further increased illumination
power. Moreover, the development of LED light sources with corresponding long pass
emission filters that are suitable for the detection of ZyOrange labeled probes would
enable the combined analysis of both cerb-B2 gene and corresponding chromosome 17
as the most reliable determination of the Her-2/neu status in patients with breast
cancer. In addition, the possibility to simply switch between different LED modules
instead of the necessity to exchange the whole elements would considerably simplify
the applicability of LED elements, since fluorescence based assays such as FISH using
commercially available dual color labeled kits are increasingly introduced into clinical
research. In summary, although there is still need for some further developments,
our own experiences emphasize the high potential of these monochromatic LED elements
with all their characteristic features to provide a serious alternative to conventional
advanced fluorescence microscopy in routine pathology. Without dispute this low-cost
technology has initiated a sustainable technological progress giving direction also
to the growing field of molecular pathology.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

DSL carried out the FISH analyses and drafted the manuscript. TZ was responsible for
the surgical part and clinical data. PZ provided the technological capabilities. HS
and EV were responsible for the histopathological aspects. TG conceived of the study
and was involved in drafting the manuscript. All authors have read and approved the
final manuscript.

Acknowledgements

The authors like to thank Dr. N. Stumpp from MEDAC for providing the VIS LED cassettes
and long pass filters for testings as well as Maria Lammers and Jasmin Tiebach for
excellent technical support.